Synaptic vesicles are distinct organelles that require multiple integral protein components to be functional. Despite this complexity, following exocytosis, synaptic vesicles are retrieved, recycled and reused swiftly within several seconds with high fidelity. The exact mechanisms that underlie this process remain poorly understood. In this renewal application, we propose to investigate mechanisms underlying synaptic vesicle exocytosis-endocytosis process by visualizing the fusion and retrieval of single synaptic vesicles using fluorescence imaging methodologies we developed in the last five years. In our hands, this imaging approach has become rather routine and versatile, amenable to molecular manipulations as well as multimodal imaging at physiological temperatures with high temporal resolution. Monitoring exo-endocytosis of single synaptic vesicles enables us to dissect permissive and instructive signals that regulate synaptic vesicle retrieval. Using this approach, our recent experiments suggested a limited role for the classical endocytosis machinery, while implying a critical role for the exocytotic fusion machinery, in regulation of quantal single vesicle endocytosis. In this renewal application, we will fully expand these initial findings and interrogate the mechanisms underlying single synaptic vesicle retrieval at physiological temperatures with high temporal resolution. To achieve this goal, we propose three specific aims. In the first aim, we will study the role of dynamins in single synaptic vesicle retrieval. The second aim will focus on the role of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) fusion machinery in single synaptic vesicle retrieval. Finally, the third aim will investigate the roles of endosomal SNAREs in spontaneous synaptic vesicle retrieval. Collectively, these complementary experiments will elucidate the molecular mechanisms ensuring the fidelity and the time course of synaptic vesicle retrieval in central synapses. Information attained from these studies will provide new insight to the synaptic substrates that may be affected by a number of in neuropsychiatric and neurological disorders including major depressive disorder, autism and schizophrenia.